Sumbalova 2014 Abstract MiP2014
|Optimization of malate concentration for high-resolution respirometry: mitochondria from rat liver and brain.|
Complex substrate-uncoupler-inhibitor titration (SUIT)  protocols, used in high-resolution respirometry, are designed to determine respiration with CI-linked and CII-linked substrates in a single experiment. Observations on multiple types of mitochondria revealed that malate, a substrate used to fuel CI-linked respiration, inhibits CII-linked (succinate+rotenone; coupled and noncoupled) respiration at 2 mM concentration. Mutual interference between succinate and malate was already described by Harris and Manger , and the effect was attributed to accumulation of oxaloacetate, an inhibitor of succinate dehydrogenase (SDH) and fumarate [1,3].
In this study, we examined the effect of various concentrations of malate on CI-linked and CII-linked respiration, as well as on the involvement of SDH as a constitutive part of Krebs cycle, in the respiration with CI-linked substrate combinations, in mitochondria from rat liver and brain. In both liver and brain mitochondria, 0.5 mM malate, added in combination with 5 mM pyruvate and 10 mM glutamate, supported >90% of maximal CI-linked respiration observed with saturating 2 mM malate. Conversely, when malate was added to noncoupled mitochondria fueled by CII-linked substrate succinate+rotenone, it inhibited ETS capacity by 6% and 8% at 0.5 mM, 22% and 25% at 2 mM, and 33% and 37% at 5 mM malate concentration, in mitochondria from liver and brain, respectively. A similar degree of inhibition of noncoupled CII-linked respiration by malate was observed when mitochondria were previously exposed to CI+II–linked substrates followed by inhibition of CI with rotenone. Assuming that this effect is caused by an indirect impact of malate on SDH, lower inhibition of respiration at lower malate concentration would be suggestive of a higher involvement of SDH in respiration with CI-linked substrate combinations. Indeed, this involvement, as determined by inhibition of SDH with 5 mM malonate after ADP-stimulated respiration with pyruvate+glutamate+malate, amounted to more than 50% without malate, ~35% with 0.5 mM, ~20% with 2 mM, and ~15% with 5 mM malate concentration in both types of mitochondria.
In summary, our observations showed that despite tremendous physiological differences between liver and brain mitochondria, malate affected their respiratory patterns in a similar manner, suggesting that this may be a more general phenomenon. Therefore, we recommend that when malate is used in complex SUIT protocols, a concentration of 0.5 mM should be used rather than previously applied higher concentrations (2 mM), balancing its stimulatory and inhibitory effects on mitochondrial respiration.
Labels: MiParea: Respiration, Instruments;methods
Organism: Rat Tissue;cell: Nervous system, Liver Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property.
Regulation: Inhibitor, Substrate, Uncoupler Coupling state: OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
1-Pharmacobiochem Lab, Fac Medicine, Comenius Univ, Bratislava, Slovakia; 2-OROBOROS INSTRUMENTS, Innsbruck, Austria; 3-Dep Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. – [email protected]
- Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp.
- Harris EJ, Manger JR (1968) Intramitochondrial substrate concentration as a factor controlling metabolism. The role of interanion competition. Biochem J 109: 239-46.
- Gunter TE, Gerstner B, Lester T, Wojtovich AP, Malecki J, Swarts SG, Brookes PS, Gavin CE, Gunter KK (2010) An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using State 3 oxidation rate assays. Toxicol Appl Pharmacol 249: 65-75.